Liquid biopsy has recently emerged as an important tool in clinical practice particularly for lung cancer patients. We retrospectively evaluated cell-free DNA analyses performed at our Institution by next generation sequencing methodology detecting the major classes of genetic alterations. Starting from the graphical representation of chromosomal alterations provided by the analysis software, we developed a support vector machine classifier to automatically classify chromosomal profiles as stable (SCP) or unstable (UCP). High concordance was found between our binary classification and tumor fraction evaluation performed using shallow whole genome sequencing. Among clinical features, UCP patients were more likely to have ≥ 3 metastatic sites and liver metastases. Longitudinal assessment of chromosomal profiles in 33 patients with lung cancer receiving immune checkpoint inhibitors (ICIs) showed that only patients that experienced early death or hyperprogressive disease retained or acquired an UCP within 3 weeks from the beginning of ICIs. UCP was not observed following ICIs among patients that experienced progressive disease or clinical benefit. In conclusion, our binary classification, applied to whole copy number alteration profiles, could be useful for clinical risk stratification during systemic treatment for non-small cell lung cancer patients.

BACKGROUND: Parathyroid cancer has been linked to germline mutations of the CDC73 gene. However, carriers harboring cancer-associated germline CDC73 mutations may develop only parathyroid adenoma or no parathyroid disease. This incomplete penetrance indicates that additional genomic events are required for parathyroid tumorigenesis.
OBJECTIVE: (1) Determine the status of the second CDC73 allele in parathyroid tumors harboring germline CDC73 mutations, and (2) compare the genomic landscapes between parathyroid carcinomas and adenomas.
METHODS: Whole-exome and RNA sequencing of 12 parathyroid tumors harboring germline CDC73 mutations (6 adenomas and 6 carcinomas) and their matched normal tissues.
RESULTS: All 12 parathyroid tumors had gained one somatic event predicted to cause a complete inactivation of the second CDC73 allele. Several distinctive genomic features were identified in parathyroid carcinomas compared to adenomas, including more single nucleotide variants bearing the C>G transversion and APOBEC deamination signatures, frequent mutations of the genes involved in the PI-3K/mTOR signaling, a greater number of copy number variations, and substantially more genes with altered expression. Parathyroid carcinomas also share some genomic features with adenomas. For instance, both have recurrent somatic mutations and copy number loss that impact the genes involved in T-cell receptor signaling and tumor antigen presentation, suggesting a shared strategy to evade immune surveillance.
CONCLUSIONS: Biallelic inactivation of CDC73 is essential for parathyroid tumorigenesis in carriers harboring germline mutations of this gene. Despite sharing some genomic features with adenomas, parathyroid carcinomas have more distinctive alterations in the genome, some of which may be critical for cancer formation.

OBJECTIVE: To perform a molecular profiling of the metastases from papillary thyroid carcinomas (PTCs) and poorly differentiated thyroid carcinomas (PDTCs).
METHODS: We retrieved and analyzed the molecular and clinical features of 136 metastases from PTCs and 35 metastases from PDTCs subjected to targeted DNA sequencing, from cBioPortal. The clinicopathological data included the number and location of the metastases, and genomic data included mutations, translocations, copy number alterations and fraction of the genome altered (FGA).
RESULTS: Bone metastases from PTCs had a lower frequency of BRAF mutations than the lymph node metastases (LNMs) (43% vs 88%, p < 0.01), and a higher frequency of RBM10 and NRAS mutations than the LNMs (21% vs 3% for both, p < 0.05). The FGA of the bone metastases was higher than the FGA of the lung metastases (5.6% vs 1.3%, p < 0.05). The frequency of RET translocations was higher in the lung metastases from PTCs than the LNMs (15% vs 3%, p < 0.05). The LNMs from PTC patients harboring 4 or more distant metastases (DMs) had a higher frequency of TERT promoter mutations than the LNMs from patients harboring less than 4 DMs (96% vs 65%, p < 0.001). SDHA gene amplifications were enriched in the bone metastases from PDTCs and absent in the LNMs (38% vs 0%, p < 0.05).
CONCLUSIONS: Metastases from PTCs and PDTCs harbor clinically relevant alterations affecting distinct body locations, such as NRAS and RBM10 mutations, RET translocations and SDHA amplifications that may be explored therapeutically.

Many cancers display whole chromosome instability (W-CIN) and structural chromosomal instability (S-CIN), referring to increased rates of acquiring numerically and structurally abnormal chromosome changes. This protocol provides detailed steps to analyze the W-CIN and S-CIN across cancer types, intending to leverage large-scale bulk sequencing and SNP array data complemented with the computational models to gain a better understanding of W-CIN and S-CIN.

The identification of recurrent genomic alterations in tumour cells has a significant role in the classification of mature B- and T-cell lymphomas. Following the development of new technologies, such as next generation sequencing and the improvement of classical technologies such as conventional and molecular cytogenetics, a huge catalogue of genomic alterations in lymphoid neoplasms has been established. These alterations are relevant to refine the taxonomy of the classification of lymphomas, to scrutinize the differential diagnosis within different lymphoma entities and to help assessing the prognosis and clinical management of the patients. Consequently, here we describe the key genetic alterations relevant in mature B- and T-cell lymphomas.

Breast cancer (BCa) is a prevalent malignancy that predominantly affects women around the world. Somatic copy number alterations (CNAs) are tumor-specific amplifications or deletions of DNA segments that often drive BCa development and therapy resistance. Hence, the complex patterns of CNAs complement BCa classification systems. In addition, understanding the precise contributions of CNAs is essential for tailoring personalized treatment approaches. This review highlights how tumor evolution drives the acquisition of CNAs, which in turn shape the genomic landscapes of BCas. It also discusses advanced methodologies for identifying recurrent CNAs, studying CNAs in BCa and their clinical impact.

Copy number alterations (CNAs) are significant in tumor initiation and progression. Identifying these aberrations is crucial for targeted therapies and personalized cancer diagnostics. Next-generation sequencing (NGS) methods present advantages in scalability and cost-effectiveness, surpassing limitations associated with reference assemblies and probe capacities in traditional laboratory approaches. This retrospective study evaluated CNAs in 50 FFPE tumor samples (breast cancer, ovarian carcinoma, pancreatic cancer, melanoma, and prostate carcinoma) using Illumina\'s TruSight Oncology 500 (TSO500) and the Affymetrix Oncoscan Molecular Inversion Probe (OS-MIP) (ThermoFisher Scientific, Waltham, MA, USA). NGS analysis with the NxClinical 6.2 software demonstrated a high sensitivity and specificity (100%) for CNA detection, with a complete concordance rate as compared to the OS-MIP. All 54 known CNAs were identified by NGS, with gains being the most prevalent (63%). Notable CNAs were observed in MYC (18%), TP53 (12%), BRAF (8%), PIK3CA, EGFR, and FGFR1 (6%) genes. The diagnostic parameters exhibited high accuracy, including a positive predictive value, negative predictive value, and overall diagnostic accuracy. This study underscores NxClinical as a reliable software for identifying clinically relevant gene alterations using NGS TSO500, offering valuable insights for personalized cancer treatment strategies based on CNA analysis.

OBJECTIVE: New prognostic markers are needed to identify patients with hepatocellular carcinoma (HCC) who carry a worse prognosis. Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess both circulating tumor DNA (ctDNA) fraction and large structural genomic alterations. Here, we studied the performance of ULP-WGS of plasma cfDNA to infer prognosis in patients with HCC.
METHODS: Plasma samples were obtained from patients with HCC prior to surgery, locoregional or systemic therapy, and were analyzed by ULP-WGS of cfDNA to an average genome-wide fold coverage of 0.3x. ctDNA and copy number alterations (CNA) were estimated using the software package ichorCNA.
RESULTS: Samples were obtained from 73 HCC patients at different BCLC stages (BCLC 0/A: n=37, 50.7%; BCLC B/C: n=36, 49.3%). ctDNA was detected in 18 out of 31 patients who received systemic treatment. Patients with detectable ctDNA showed significantly worse overall survival (median, 13.96 months vs not reached). ctDNA remained an independent predictor of prognosis after adjustment by clinical-pathologic features and type of systemic treatment (hazard ratio 7.69; 95%, CI 2.09-28.27). Among ctDNA-positive patients under systemic treatments, the loss of large genomic regions in 5q and 16q arms was associated with worse prognosis after multivariate analysis.
CONCLUSIONS: ULP-WGS of cfDNA provides clinically relevant information about the tumor biology. The presence of ctDNA and the loss of 5q and 16q arms in ctDNA-positive patients are independent predictors of worse prognosis in patients with advanced HCC receiving systemic therapy.

The genetic repertoire of primary thyroid cancers (TCs) is well documented, but there is a considerable lack of molecular profiling in metastatic TCs. Here, we retrieved and analyzed the molecular and clinical features of 475 primary and metastatic TCs subjected to targeted DNA sequencing, from the cBioPortal database. The cohort included primary and metastatic samples from 276 papillary thyroid carcinomas (PTCs), 5 follicular thyroid carcinomas, 22 Hürthle cell carcinomas (HCCs), 127 poorly differentiated thyroid carcinomas (PDTCs), 30 anaplastic thyroid carcinomas (ATCs) and 15 medullary thyroid carcinomas. The ATCs had the highest tumor mutational burden and the HCCs the highest fraction of the genome altered. Compared to primary PTCs, the metastases had a significantly higher frequency of genetic alterations affecting TERT (51% vs 77%, P < 0.001), CDKN2A (2% vs 10%, P < 0.01), RET (2% vs 7%, P < 0.05), CDKN2B (1% vs 6%, P < 0.05) and BCOR (0% vs 4%, P < 0.05). The distant metastases had a significantly lower frequency of BRAF (64% vs 85%, P < 0.01) and a significantly higher frequency of NRAS (13% vs 3%, P < 0.05) hotspot mutations than the lymph node metastases. Metastases from HCCs and PDTCs were found to be enriched for NF1 (29%) and TP53 (18%) biallelic alterations, respectively. The frequency of subclonal mutations in ATCs was significantly higher than in PTCs (43% vs 25%, P < 0.01) and PDTCs (43% vs 22%, P < 0.01). Metastatic TCs are enriched in clinically informative genetic alterations such as RET translocations, BRAF hotspot mutations and NF1 biallelic losses that may be explored therapeutically.

Adult acute lymphoblastic leukemia (ALL) is associated with poor outcomes. ALL is initiated by primary aberrations, but secondary genetic lesions are necessary for overt ALL. In this study, we reassessed the value of primary and secondary aberrations in intensively treated ALL patients in relation to mutator enzyme expression. RT-PCR, genomic PCR, and sequencing were applied to evaluate primary aberrations, while qPCR was used to measure the expression of RAG and AID mutator enzymes in 166 adult ALL patients. Secondary copy number alterations (CNA) were studied in 94 cases by MLPA assay. Primary aberrations alone stratified 30% of the patients (27% high-risk, 3% low-risk cases). The remaining 70% intermediate-risk patients included BCR::ABL1pos subgroup and ALL lacking identified genetic markers (NEG ALL). We identified three CNA profiles: high-risk bad-CNA (CNAhigh/IKZF1pos), low-risk good-CNA (all other CNAs), and intermediate-risk CNAneg. Furthermore, based on RAG/AID expression, we report possible mechanisms underlying the CNA profiles associated with poor outcome: AID stratified outcome in CNAneg, which accompanied most likely a particular profile of single nucleotide variations, while RAG in CNApos increased the odds for CNAhigh/IKZF1pos development. Finally, we integrated primary genetic aberrations with CNA to propose a revised risk stratification code, which allowed us to stratify 75% of BCR::ABL1pos and NEG patients.